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目的研究RhoA蛋白在内皮细胞迁移过程中的作用。方法采用脂质体包绕法将RhoA野生型(RhoAwt)、稳定表达持续活化型(RhoA63L)、主导抑制型(RhoA188A)突变质粒转染进入血管内皮细胞,获得稳定表达的EA.hy926-RhoAwt、EA.hy926-RhoA63L和EA.hy926-RhoA188A细胞。然后,利用Westernblot检测细胞中活性RhoA蛋白含量。最后,利用化学趋化因子IL-8(100ng/ml)分别刺激这3组细胞和对照组细胞,观察细胞迁移情况,并分析细胞迁移与活性RhoA之间的关系。结果1)各组细胞活性RhoA含量较对照组均有变化,EA.hy926-RhoAwt组稍稍升高,EA.hy926-RhoA63L组明显升高,而EA.hy926-RhoA188A组明显降低;2)在IL-8刺激下,EA.hy926-RhoAwt组细胞迁移能力稍高于对照组,EA.hy926-RhoA63L组细胞迁移能力远高于对照组,而EA.hy926-RhoA188A组细胞迁移能力则被明显抑制。因此,在IL-8刺激下,各组细胞迁移能力与细胞活性RhoA蛋白含量正相关。结论RhoA蛋白是IL-8诱导内皮细胞迁移过程中的关键分子,这有助于我们进一步探讨IL-8诱导内皮细胞迁移的理论机制和临床应用。
Objective To study the role of RhoA in endothelial cell migration. Methods RhoA wt, RhoA63L and RhoA188A mutant plasmids were transfected into vascular endothelial cells by lipofectamine to obtain stable expression of EA.hy926-RhoAwt, EA.hy926-RhoA63L and EA.hy926-RhoA188A cells. Then, the amount of active RhoA protein in the cells was detected by Western blot. Finally, the chemotactic factor IL-8 (100 ng / ml) was used to stimulate the three groups of cells and the control group respectively to observe the cell migration and to analyze the relationship between cell migration and active RhoA. The results showed that: 1) The content of RhoA in cells in each group changed slightly compared with that in the control group. EA.hy926-RhoAwt group was slightly increased, EA.hy926-RhoA63L group was significantly increased, and EA.hy926-RhoA188A group was significantly reduced; 2) The migration ability of EA.hy926-RhoA188A group was significantly higher than that of the control group, while the migration ability of EA.hy926-RhoA63L group was much higher than that of the control group, while the migration ability of EA.hy926-RhoA188A group was significantly inhibited. Therefore, under the stimulation of IL-8, the migration ability of cells in each group was positively correlated with the content of cellular RhoA protein. Conclusion RhoA protein is a key molecule in the process of endothelial cell migration induced by IL-8, which will help us to further explore the theoretical mechanism and clinical application of IL-8 induced endothelial cell migration.